Browsing by Author "JABALQUINTO, AM"

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    CHEMICAL MODIFICATION OF ESSENTIAL ARGININES IN PIG-LIVER PHOSPHOMEVALONATE KINASE
    (1982) VERGARA, M; ALVEAR, M; CARDEMIL, E; JABALQUINTO, AM; EYZAGUIRRE, J
    Phosphomevalonate [MVAP] kinase, an enzyme of the polyisoprenoid biosynthesis pathway, catalyzes the transfer of phosphate from ATP to MVAP, with the formation of pyrophosphomevalonate and ADP. The pig liver enzyme, a monomer of MW 22,000, possesses 1 cysteinyl residue, which is essential for catalysis. By means of chemical modification of a partially purified preparation, the participation of arginine residues in the enzyme active site was studied. Butanedione and phenylglyoxal were chosen as group-specific reagents. The kinetics of inactivation by both reagents is rather complex, suggesting that several arginine residues, directly or indirectly related to the active site, are being modified. Both substrates, MVAP and Mg-ATP, protect against inactivation but to a different extent, depending on the modifier used. With butanedione, almost total protection is achieved with Mg-ATP. Better protection with MVAP is observed for the modification with phenylglyoxal, but with this reagent, Mg-ATP protects strongly only at very high concentrations. A reversible inactivation mechanism is followed with butanedione, while for phenylglyoxal this mechanism appears to be irreversible, in agreement with findings by other authors. MVAP kinase probably presents 2 or more arginyl residues in or near its active site, one of them being involved in the binding of Mg-ATP, and at least another located in the neighborhood of the MVAP binding site. A more precise determination of the number of essential residues requires its measurement by chemical methods, utilizing a homogeneous enzyme preparation.
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    PIG-LIVER PHOSPHOMEVALONATE KINASE .1. PURIFICATION AND PROPERTIES
    (1980) BAZAES, S; BEYTIA, E; JABALQUINTO, AM; SOLISDEOVANDO, F; GOMEZ, I; EYZAGUIRRE, J
    Pig liver phosphomevalonate kinase (EC 2.7.4.2) was purified to homogeneity as shown by polyacrylamide gel electrophoresis. The MW estimates range from 21,000 to 22,500. Each molecule is composed of 1 polypeptide chain. The presence of SH-containing reagents is essential for preservation of enzyme activity at all steps in the purification. The enzyme shows absolute specificity for ATP and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 7.5 to over 9.5. Kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.075 mM and 0.46 mM were obtained for phosphomevalonate and ATP, respectively. Amino acid composition shows a high content of acid amino acids, 1 cysteine residue/molecule of enzyme, and the absence of methionine. The enzyme apparently plays no regulatory function is cholesterol biosynthesis in pig liver, although a variable enzyme content was detected in different livers.
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    PIG-LIVER PHOSPHOMEVALONATE KINASE .2. PARTICIPATION OF CYSTEINYL AND LYSYL GROUPS IN CATALYSIS
    (1980) BAZAES, S; BEYTIA, E; JABALQUINTO, AM; SOLISDEOVANDO, F; GOMEZ, I; EYZAGUIRRE, J
    Phosphomevalonate kinase from pig liver is inactivated by 5,5''-dithiobis(2-nitrobenzoate) and pyridoxal 5''-phosphate. The substrate phosphomevalonate protects the enzyme against inactivation by these reagents. Inactivation by 5,5''-dithiobis(2-nitrobenzoate) is complete and may be reverted by 2-mercaptoethanol or dithiothreitol. Experiments carried out with partially inactivated enzyme show no change in the kcat or in the apparent Km for the substrates, as compared with the native enzyme, indicating the existence of 2 populations of molecules, one intact and the other totally inactive. 5,5''-Dithiobis(2-nitrobenzoate) apparently reacts with the only cysteinyl residue of the enzyme and this residue is located in or near the active site. Inhibition by pyridoxal 5''-phosphate can be reverted, either by dialysis or by the addition of lysine, but not if the partially inactivated enzyme is treated previously with NaBH4, in agreement with the formation of a Schiff base between pyridoxal 5''-phosphate and an amino group of the enzyme. This is further supported by the appearance of an absorption band with a maximum at 325 nm in the enzyme treated with pyridoxal 5''-phosphate and NaBH4. Pyridoxal and pyridoxamine 5''-phosphate are weaker inhibitors than pyridoxal 5''-phosphate, suggesting a specific effect due to the phosphate and aldehyde groups. The enzyme is not completely inactivated by pyridoxal 5''-phosphate, even at a molar ratio of 350, or by a 2nd inactivation treatment after reduction with NaBH4. The partially modified enzyme shows a lower Km for phosphomevalonate than the native enzyme, suggesting that the reactive group is located near the binding site of phosphomevalonate. The lower Km may reflect an effect of the positive charge of the pyridoxal 5''-phosphate ring N, enhancing the binding of phosphomevalonate. Values of 8.15 at 24.degree. C and 7.95 at 31.degree. C were determined for the pK of the reactive group. A .DELTA.Hi of 11.8 kcal/mol was estimated, in agreement with the values expected for an amino group. One amino group/active site is involved in the enzyme inactivation as shown by kinetic data. Quantification of the number of moles of pyridoxal 5''-phosphate bound/mole of enzyme is not conclusive but supports this assertion. This group may correspond to an .epsilon.-amino group of lysine.
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    POSSIBLE INVOLVEMENT OF CYSTEINYL, LYSYL AND ARGINYL GROUPS IN THE ACTIVE-CENTER OF PIG-LIVER PHOSPHOMEVALONATE KINASE
    (1981) BAZAES, S; BEYTIA, E; JABALQUINTO, AM; DEOVANDO, FS; GOMEZ, I; EYZAGUIRRE, J
    Pig liver phosphomevalonate kinase has a MW of about 22,000, its structure is monomeric and it shows only 1 cysteine residue/molecule. The presence of cysteinyl and lysyl residues at the active site was studied by chemical modification. Preliminary work was also performed on the involvement of arginyl residues. Modification of cysteinyl residues was attempted using 5,5''dithiobis (2-notrobenzoate) (DTNB). Pyridoxal phosphate (PLP) was utilized as reagent for lysyl residues, and arginyl residues were modified with butanedione. Inactivation of the enzyme by DTNB is complete but may be reversed by 2-mercaptoethanol or dithiothreitol. Kinetic analysis shows that 1 mol of reactive SH group/mol of enzyme is responsible for catalytic activity. The substrate phosphomevalonate, but not Mg-ATP, protects against inactivation. Since no change in Km or kcat is observed on partial inactivation, this is interpreted as indication that the modified molecule is totally inactive. PLP inactivates the enzyme, but even at high concentrations total inactivation was not observed. Addition of lysine or dialysis reactivates the modified enzyme, but this does not occur after the addition of sodium borohydride. Pyridoxal and pyridoxamine 5''-phosphate are weaker inhibitors than PLP. As with DTNB, phosphomevalonate but not MgADP protects against inactivation. The partially modified enzyme shows a lower Km for phosphomevalonate than the native enzyme. Kinetic data suggest that 1 PLP molecule reacts/enzyme active center, while spectrophotometric titration shows a lower value. pK values 7.2 (at 24.degree. C) and 7.5 (at 31.degree. C) are obtained for the modified group, in agreement with values described for reactive lysines. Inactivation by butanedione can be protected by phosphomevalonate, but only to a smaller extent by Mg-ATP. The cysteinyl residue of the enzyme plays an essential role in the catalytic mechanism, while the lysine residue modified may not be essential for activity. Both residues, and possibly an arginyl group, may be located at or near the phosphomevalonate binding site of the enzyme.
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    PURIFICATION AND CHARACTERIZATION OF AVIAN LIVER MEVALONATE-5-PYROPHOSPHATE DECARBOXYLASE
    (1982) ALVEAR, M; JABALQUINTO, AM; EYZAGUIRRE, J; CARDEMIL, E
    Mevalonate-5-pyrophosphate decarboxylase was purified 5800 times from chicken liver and obtained in a stable and highly purified form. The protein is a dimer of MW 85,400 .+-. 1941, and its subunits were not resolved by gel electrophoresis in denaturing conditions. The purified enzyme does not require the presence of SH-containing reagents for either activity or stability. The enzyme shows a high specificity for ATP and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 4.0-6.5. Inhibitory effects for the enzyme activity were detected by citrate, phthalate, and phosphate. The isoelectric point, as determined by column choromatofocusing, is 4.8. The kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.0141 mM and 0.504 mM were obtained for mevalonate-5-pyrophosphate and ATP, respectively.